CN110066343A - A kind of recombinant antigen and its application for detecting HIV new infections - Google Patents
A kind of recombinant antigen and its application for detecting HIV new infections Download PDFInfo
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- CN110066343A CN110066343A CN201910433891.2A CN201910433891A CN110066343A CN 110066343 A CN110066343 A CN 110066343A CN 201910433891 A CN201910433891 A CN 201910433891A CN 110066343 A CN110066343 A CN 110066343A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
The recombinant antigen and its application that the invention discloses a kind of for detecting HIV new infections, it is related to antigen preparation technical field, recombinant antigen includes B amino acid sequence, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation, hydrophilic amino acid core sequence and the lysine sequence of antigen both ends addition, B amino acid sequence is as shown in SEQ ID No.1, E amino acid sequence is as shown in SEQ ID No.2, D amino acid sequence is as shown in SEQ ID No.3, hydrophilic amino acid core sequence is as shown in SEQ ID No.4, BC series of variation is as shown in SEQ ID No.5, AE series of variation is as shown in SEQ ID No.6.The solubility of the recombinant antigen is high, is easy to purify, is easy to mark, and erroneous judgement caused by the false positive reaction and hypotype when being avoided that or reduce HIV detection new infections are omitted improves the accuracy of detection.
Description
Technical field
The present invention relates to antigen preparation technical fields, in particular to a kind of for detecting the recombination of HIV new infections
Antigen and its application.
Background technique
AIDS (Human immunodeficiency virus, HIV) seriously threatens human health, is that the whole world is common
The great public health problem faced." AIDS Epidemic report " display of The Joint Programme on AIDS publication, whole world survival
Patients infected hiv and patient up to 34,000,000 people, annual 2500000 people of new infections, dead 1,700,000 people, AIDS is anti-
It is still severe to control situation.
Currently, the infection population of AIDS spreads through sex intercourse in diversification and has become the main path of aids transmission, simultaneously
Intravenous drug use secondhand injectors, unlicensed prostitute crowd's condom utilization rate is lower and the risk factors such as male same sexual behaviour are widely present,
So that Prevention & Control of AIDS work still faces huge challenge.
The detection of AIDS virus new infections is the important content of AIDS Surveillance work, for understanding HIV spread in time
Dynamically, people at highest risk is found, assessment control effect is of great significance.In recent years, with the development of biotechnology, so that utilizing
Laboratory method judgement is infected recently, and then estimates that disease incidence becomes research hotspot.
But the antigen research currently used for the detection of AIDS virus new infections is seldom, and false sun rate is high.
Summary of the invention
The first object of the present invention is to provide a kind of for detecting the recombinant antigen of HIV new infections, the recombinant antigen
Solubility it is high, be easy to purify, can be avoided or reduces the false positive reaction occurred when the detection of HIV new infections, improve and detect
Sensitivity and accuracy.
The second object of the present invention is to provide a kind of nucleic acid point of recombinant antigen for code detection HIV new infections
Son can encode a kind of recombinant antigen suitable for detecting the detection of AIDS virus new infections, and the recombinant antigen has
The high advantage of Detection accuracy.
The third object of the present invention is to provide a kind of expression vector comprising above-mentioned coding is for detecting AIDS virus
The nucleic acid molecules of the recombinant antigen of new infections detection.
The fourth object of the present invention is to provide a kind of expression bacterial strain comprising has above-mentioned new for detecting AIDS virus
The expression vector of the recombinant antigen of hair infection detection.
The fifth object of the present invention is to provide a kind of for detecting the detection kit of HIV new infections, detection examination
Agent box can accurately detect AIDS virus new infections.
The present invention is implemented as follows:
The embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections comprising B amino acid sequence
Column, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, it is described
Hydrophilic amino acid core sequence is set between B, E, D amino acid sequence, BC and AE series of variation；
The B amino acid sequence is as shown in SEQ ID No.1, and the E amino acid sequence is as shown in SEQ ID No.2, institute
D amino acid sequence is stated as shown in SEQ ID No.3, the hydrophilic amino acid core sequence is described as shown in SEQ ID No.4
BC series of variation is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.
It is above-mentioned for detecting the core of the recombinant antigen of HIV new infections for encoding that the embodiment of the invention also provides a kind of
Acid molecule, the nucleic acid molecules include B nucleic acid molecules, E nucleic acid molecules, D nucleic acid molecules, BC nucleic acid molecules, AE nucleic acid molecules with
And hydrophilic nucleotide sequence；
The nucleotide sequence of the B nucleic acid molecules is as shown in SEQ ID No.8, the nucleotide sequence of E nucleic acid molecules such as SEQ
Shown in ID No.9, the nucleotide sequence of D nucleic acid molecules is as shown in SEQ ID No.10, hydrophilic nucleotide sequence such as SEQ ID
Shown in No.11, the nucleotide sequence of the BC nucleic acid molecules is as shown in SEQ ID No.12, the nucleotide of the AE nucleic acid molecules
Sequence is as shown in SEQ ID No.13.
The embodiment of the invention also provides a kind of expression vectors comprising has above-mentioned nucleic acid molecules；
Preferably, the expression vector is pET28a.
The embodiment of the invention also provides a kind of expression bacterial strains comprising has above-mentioned expression vector；
Preferably, the expression bacterial strain is E. coli expression strains；
Preferably, the expression bacterial strain is e. coli bl21 (DE3).
It is a kind of for detecting the detection kit of HIV new infections comprising above-mentioned recombinant antigen or above-mentioned nucleic acid point
Son.
The invention has the following advantages:
The embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections, the Recombinant antigen B amino acid
Sequence, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, parent
Aqueous amino acid core sequence is set between B, E, D amino acid sequence, BC and AE series of variation；B amino acid sequence such as SEQ
Shown in ID No.1, E amino acid sequence is as shown in SEQ ID No.2, and D amino acid sequence is as shown in SEQ ID No.3, hydrophily
Amino acid core sequence is as shown in SEQ ID No.4, and BC series of variation is as shown in SEQ ID No.5, and the AE series of variation is such as
Shown in SEQ ID No.6.The solubility of the recombinant antigen is high, is easy to purify, when can be avoided or reduce the detection of HIV new infections
False positive reaction, improve the sensitivity and accuracy of detection.
It include above-mentioned for detecting the nucleic acid of the recombinant antigen of HIV new infections the embodiment of the invention also includes having a kind of
Molecule, the expression vector including the nucleic acid molecules, the expression bacterial strain including the expression vector and including above-mentioned recombinant antigen or
The detection kit for being used to detect HIV new infections of nucleic acid molecules, the detection kit have the advantages that detection accuracy is high.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is that the HIV-Ag11 in the embodiment of the present invention 2 expresses SDS-PAGE electrophoresis after bacterial strain ultrasonication；
Fig. 2 is HIV-Ag11 SDS-PAGE electrophoresis after purification in the embodiment of the present invention 3.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Below specifically to the recombinant antigen for being used to detect HIV new infections in the embodiment of the present invention and its using progress
It is bright.
The laboratory testing method of new infections, can be there are many method according to the marker difference of detection.Serology
Method before sun turns mainly has viral nucleic acid (Ribonucleic acid, RNA) detection and the detection of p24 antigen；Serology sun turns
Afterwards, according to testing principle difference, be roughly divided into 6 classes: antibody titer detection, affinity of antibody detection, specific antibody Zhan always resist
The ratio detection of body, is linearly immunized p24 antigen immunoglobulin 3 antibody (Immunoglobulin G3, IgG3) antibody test
Trace detection, anti-specific immunity antigenicity reaction detection.Wherein, by the original of the total antibody ratios of HIV gp41 specific antibody Zhan
The HIV-1 BED of reason exploitation captures EIA method (BED-CEIA), abbreviation BED method.
The mature antigen for being currently used for BED method is artificial chemically synthesized polypeptide, and specially artificial chemistry is respectively synthesized
B, E, D polypeptide, then 3 polypeptides are linked together with chemical coupling method, the preparation process of the polypeptide is complicated, antigen yield is low,
It is at high cost, and technique is unstable, repetition differs, difference between batch is big, brings great difficulty for the application of antigen.Gene work
Then dissolubility is poor, label is difficult for the BED antigen of journey recombination, false positive rate is high, is not used to the exploitation of BED method kit.
The embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections comprising has B amino acid sequence
Column, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, it is described
Hydrophilic amino acid core sequence is set between B, E, D amino acid sequence, BC and AE series of variation.
B amino acid sequence is as shown in SEQ ID No.1, and the E amino acid sequence is as shown in SEQ ID No.2, the D ammonia
Base acid sequence is as shown in SEQ ID No.3, and as shown in SEQ ID No.4, the BC becomes the hydrophilic amino acid core sequence
Different sequence is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.The recombinant antigen has solubility
Height, the false positive rate of detection HIV new infections is low, is easy to purify, and the recombinant antigen that joined AE series of variation and BC series of variation is suitable
For the detection of China's HIV new infections, have the advantages that detection accuracy is high.
Further, the N-terminal and C-terminal for detecting the recombinant antigen sequence of HIV new infections are added with for improving
Antigen can be markup lysine sequence.
Further, in the sequence of the recombinant antigen, the B amino acid sequence, E amino acid sequence, D amino acid sequence
Column, the BC series of variation and AE series of variation are successively arranged successively from the N-terminal of recombinant antigen to C-terminal.
It is further preferred that the sequence of the recombinant antigen for detecting HIV new infections is as shown in SEQ ID No.7.
It is above-mentioned for detecting the core of the recombinant antigen of HIV new infections for encoding that the embodiment of the invention also provides a kind of
Acid molecule, the nucleic acid molecules include B nucleic acid molecules, E nucleic acid molecules, D nucleic acid molecules, BC nucleic acid molecules and AE nucleic acid molecules with
And hydrophilic nucleotide sequence.
The nucleotide sequence of B nucleic acid molecules is as shown in SEQ ID No.8, the nucleotide sequence of E nucleic acid molecules such as SEQ ID
Shown in No.9, the nucleotide sequence of D nucleic acid molecules is as shown in SEQ ID No.10, hydrophilic nucleotide sequence such as SEQ ID No.11
Shown, the nucleotide sequence of BC nucleic acid molecules is as shown in SEQ ID No.12, the nucleotide sequence such as SEQ of the AE nucleic acid molecules
Shown in ID No.13.
Further, the nucleotide sequence of nucleic acid molecules is as shown in SEQ ID No.14.
The embodiment of the invention also provides a kind of expression vectors comprising has above-mentioned nucleic acid molecules.Preferably, the expression
Carrier is pET28a.
Further, the embodiment of the invention also provides a kind of expression bacterial strains comprising has above-mentioned expression vector.It is preferred that
Ground, the expression bacterial strain are E. coli expression strains；Preferably, the expression bacterial strain is e. coli bl21 (DE3).
The embodiment of the invention also provides a kind of for detecting the detection kit of HIV new infections comprising above-mentioned use
In the recombinant antigen of detection HIV new infections or the nucleic acid molecules of the above-mentioned recombinant antigen for detecting HIV new infections.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
It is provided in this embodiment a kind of for detecting the recombinant antigen of HIV new infections comprising B amino acid sequence, E ammonia
Base acid sequence, D amino acid sequence, BC series of variation and AE series of variation, hydrophilic amino acid core sequence and recombinant antigen N
The lysine sequence at end and C-terminal addition.
B amino acid sequence, E amino acid sequence, D amino acid sequence, the BC series of variation and AE series of variation successively from
The N-terminal of recombinant antigen is arranged successively to C-terminal.The hydrophilic amino acid core sequence be set to B, E, D amino acid sequence and
Between BC series of variation and AE series of variation.
Specifically, B amino acid sequence is as shown in SEQ ID No.1, the E amino acid sequence as shown in SEQ ID No.2,
The D amino acid sequence is as shown in SEQ ID No.3, and the hydrophilic amino acid core sequence is as shown in SEQ ID No.4；BC
Series of variation is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.
Further, be added with can be markup for improving antigen for the N-terminal of the amino acid sequence of recombinant antigen and C-terminal
Lysine sequence.
In the present embodiment, recombinant antigen is named as HIV-Ag11, the amino acid sequence of recombinant antigen such as SEQ ID No.7
It is shown.
Embodiment 2
For detecting the building of the recombinant antigen of HIV new infections
1. target gene fragment connects transformed clone bacterium
The B/E/D-pUC plasmid (B/E/D nucleic acid molecules) and BC/AE-pUC plasmid (BC/AE nucleic acid molecules) of gene chemical synthesis
Comprising recombinant antigen sequence sequence, using pET28a as expression vector, initiation codon contains His label, merges table
The albumen reached is purified using Ni column.
Specifically, B/E/D-pUC plasmid carries out double digestion, BC/AE-pUC plasmid EcoR I and Xho with Nde I and EcoR I
I digestion, pET28a use Nde I and I digestion of Xho as skeleton, and digestion system is carried out according to table 1,30 DEG C of digestion 30min.This experiment
Restriction enzyme enzyme cutting used is purchased from Beijing Quanshijin Biotechnology Co., Ltd, is the fast enzyme cutting of its Flycut series.
1 double digestion system of table
| Enzyme 1 | 1μl |
| Enzyme 2 | 1μl |
| 10×buffer | 2μl |
| Substrate | 16μl |
| Total | 20μl |
Three digestion systems all do agarose gel electrophoresis and DNA purification and recovery, and used kit is purchased from Beijing ancient cooking vessel state
Prosperous biotechnology Co., Ltd, glue recovery product are connected 16 hours by linked system shown in table 2, DNA ligase
Solution I is purchased from TAKARA, converts escherichia coli DH5a, obtains connection transformed clone bacterium, and escherichia coli DH5a competence is thin
Born of the same parents are purchased from Beijing Quanshijin Biotechnology Co., Ltd.
2 linked system of table
| Genetic fragment B/E/D | 2μl |
| Genetic fragment AE/BC | 2μl |
| pET28a | 1μl |
| SoutionⅠ | 5μl |
| Total volume | 10μl |
2. positive spot screening and plasmid extract
The plated growth of connection transformed clone bacterium in step 1 has white Escherichia coli list spot, and picking plate list spot carries out
Bacterium colony PCR, PCR primer are as follows:
pET-fw:CACTATAGGGGAATTGTGAGCGGATAAC(SEQ ID No.15)。
pET-rv:CTCAGCTTCCTTTCGGGCTTTGTTAG(SEQ ID No.16)。
Bacterium colony PCR reaction system component is shown in Table 3, PCR response procedures are as follows: initial denaturation: 94 DEG C of 5min；Circulation: 94 DEG C of 30s,
60 DEG C of 30s, 72 DEG C of 90s, totally 30 times circulation；Extend: 72 DEG C of 5min.Positive spot colony PCR product clip size theory is
1034bp。
3 bacterium colony PCR reaction system of table
| Template DNA | 0.5μl |
| pET-fw(20μM) | 1μl |
| pET-rv(20μM) | 1μl |
| 2×TaqMix | 10μl |
| ddH2O | 7.5μl |
| Total volume | 20μl |
It by the single spot of the positive screened, shakes bacterium and stays overnight, next day extracts plasmid, and plasmid extraction kit is that OMEGA plasmid is small
Extraction reagent kit.The HIV-Ag11 plasmid of acquisition, plasmid double digestion (I+Xho I of Nco) are accredited as the positive, raw work bioengineering are sent to have
The sequencing of Beijing sequencing portion, limit company, sequence and design are completely the same, can be used to conversion Bacillus coli expression bacterium.
3.HIV-Ag11 plasmid converts Bacillus coli expression bacterium BL21 (DE3)
1 μ l of HIV-Ag11 plasmid is taken, 50 μ l e. coli bl21 (DE3) competent cells are added, place 30min on ice,
42 DEG C of heat shock 90s place 2min on ice, and 500 μ l LB culture mediums (being free of antibiotic) is added, and 37 DEG C, 200rpm is cultivated 1 hour,
60ul culture solution spread plate (containing 50 μ g/ml of kanamycin sulfate) is taken, obtains including the big of recombinant antigen HIV-Ag11
Enterobacteria expresses bacterium BL21 (DE3).E. coli bl21 (DE3) competent cell used is tested purchased from the complete biological skill of formula gold in Beijing
Art Co., Ltd.
4. Escherichia coli inducing expression
The glycerol stock for expressing bacterium accesses 300ml culture medium (containing 50 μ g/ml of kanamycin sulfate), and 37 DEG C, 200rpm shakes
Bottle is cultivated to OD600=0.8, it is added IPTG inducing expression (the final concentration of 0.7mM of IPTG), continues to cultivate 4h.By bacterium solution
6500rpm is centrifuged 10min, abandons supernatant, collects bacterium mud.
5. Escherichia coli ultrasonication
10mMTris-HCl (pH8.0) is added in the ratio of 1g bacterium mud 40ml buffer in the Escherichia coli bacterium mud of expression, weight
Outstanding thallus, the ultrasonication on ultrasonic machine, 20ml bacterium solution, 200w, ultrasonic 2s stop 2s ultrasound 20min.Ultrasonic complete bacterium solution compared with
Be it is limpid, take 50 μ l ultrasound liquid, be centrifuged 10min precipitation and separation and supernatant through 11000rpm, precipitating is used as inclusion body sample.It takes
Clear 30 μ l carries out whether having purpose protein expression in SDS-PAGE detection precipitating and supernatant, please refers to attached drawing as Supernatant samples
1, HIV-Ag11 protein expression form is inclusion body expression, and inclusion body is taken to carry out follow-up test.
Embodiment 3
For detecting the purifying of the recombinant antigen of HIV new infections.
The broken thallus that 2 step 5 of embodiment is obtained is usedJ-E Beckman centrifuge is centrifuged, from
Mental and physical efforts: 22000g, temperature: 4-8 DEG C, the time: 15 minutes.Supernatant is abandoned, inclusion body precipitating is retained.
By inclusion body with by precipitating inclusion body lysate (10mM Tris-HCl+500mM NaCl pH 8.0+8mM
Urea it) dissolves, after inclusion body cracks completely, 22000g, centrifugation 15min removes impurity, retains supernatant, i.e. Ni Sepharose
Fast Flow column chromatography samples.
The Ni Sepharose Fast Flow gel content of suitable chromatographic column and filling is selected to carry out dress column.It will fill
Ni Sepharose Fast Flow column chromatography water afterwards rinses 4 column volumes.With the equilibrium liquid (10mM of 4 times of column volumes
Tris-HCl+500mM NaCl pH 8.0+8mM Urea) balance purification column, loading after the completion of balance.After end of the sample, with flat
Remaining sample in the liquid flushing pipeline that weighs and chromatographic column.With removal of impurities liquid (the 10mM Tris-HCl pH 8.0+500mM of 4 times of column volumes
NaCl+30mM IMZ+8mM Urea) elution removing impurity.With eluent (the 10mM Tris-HCl pH 8.0+ of 4 times of column volumes
500mM NaCl+200mM IMZ+8mM Urea) elution of bound antigen protein, with the CB of 50mM pH9.6 dialysis removal denaturation
After agent and imidazoles, recombinant antigen as after purification.
This test is gone back while being constructed the antigen HIV-BED of BED polypeptide expressing in series, expression vector pET28a, expression
Bacterium is BL21 (DE3), and protein expression form is also inclusion body expression.Use inclusion body lysate (10mM Tris-HCl+500mM
NaCl pH 8.0+8mM Urea) dissolution, inclusion body cracking is incomplete, after taking the hydrotropies measures such as 42 DEG C of processing, ripples ultrasounds
It can not still crack completely.After its lysate is centrifuged removal precipitating, there are HIV-BED albumen, use and HIV-Ag11 in supernatant
Identical method (Ni column) is purified.SDS-PAGE detection is carried out after purification, and testing result please refers to attached drawing 2 and table 4.
4 HIV-Ag11 and HIV-BED inclusion body of table cracks situation comparison
By table 4 and Fig. 2 it is found that for the HIV-BED antigen of the recombinant antigen that provides of embodiment 1 compared with the existing technology,
More easily cracking purification.
Embodiment 4
The specificity and Detection of Stability for the recombinant antigen for detecting HIV new infections that embodiment 3 provides.
Goat anti-human igg is coated with 96 orifice plates, enzyme combination diluent, A liquid, B liquid, terminate liquid and is purchased from Ying Kexin wound (Xiamen)
Science and Technology Ltd..
Fused antigen label horseradish peroxidase (Over-voltage protection): weighing 10mgHRP and be dissolved in 1ml ultrapure water, room
The lower stirring of temperature dissolves it sufficiently, and the 0.1M NaIO that 0.2ml newly matches is added in upper liquid4Solution is protected from light stirring 25 minutes；Oxygen
The ethylene glycol solution after 0.2ml dilution is added after the completion of change in upper liquid, continues to be protected from light stirring 30 minutes；Above-mentioned reaction solution is added
In HIV-Ag11 protein solution after to dialysis, dialysis 4.5h is carried out with 50mM pH9.5 carbonate buffer solution after mixing, it is intermediate
It changes the liquid once；The 10mg/ml NaBH that 0.4ml newly matches is added4Solution mixes, then sets 4 DEG C of 2h；Above-mentioned liquid is packed into bag filter
In, it is dialysed with 20mM TBS pH8.0 buffer, at room temperature overnight, liquid is changed twice in centre；Next day adds into obtained finished product enzyme
The glycerol of equivalent mixes, -20 DEG C of preservations.
Enzyme-labelled antigen Activity determination:
Required amount of capillary strip for being coated with goat anti-human igg is taken, is placed on grillage, and carries out mark.Distinguish in order
100 μ l key quality-control products and positive and negative control are added in corresponding aperture.It sets in 37 DEG C of insulating boxs and incubates 60min, filled with cleaning solution
Divide washing 5 times, then detains dry (cleaning solution soaking time 30-60 seconds).
100 μ l enzyme-labelled antigens (1:5000 dilution) is added in every hole respectively, is placed in 37 DEG C of insulating boxs and incubates 30min
(can onboard cover sealing plate film if needed) is sufficiently washed 5 times with cleaning solution, and then button is dry.It is each that substrate A, B liquid is added in every hole
50 μ l after patting mixing, are placed in 37 DEG C of insulating boxs and incubate 30min.50 μ l of terminate liquid is added in every hole, mixes.With microplate reader list
Wavelength 450nm or dual wavelength 450/630nm measure each hole A value (when being measured with Single wavelength, need to use blank control air-conditioning zero, one
As use dual wavelength method), and record result.
UtoffValue (COV) is calculated: (negative control OD value is lower than 0.075 for COV=negative control mean OD value × 2.0
Make 0.075 to calculate, be higher than 0.075 and calculated by practical OD value), sample to be tested OD value >=COV is the positive, sample to be tested OD value <
COV is feminine gender.
Specific detection:
The HIV positive serum of confirmation 112, negative serum 1000 are detected by enzyme-labelled antigen detection method.
The HIV-Ag11 that embodiment 3 provides also carries out horseradish peroxidase-labeled using identical method, and detects work
Property and specificity.Testing result please refers to table 5 and table 6.
5 enzyme-labelled antigen Activity determination result of table
6 specific detection result of table
By table 5 and table 6 it is found that the antigen active and specificity of the recombinant antigen HIV-Ag11 that embodiment 3 provides are superior to
The HIV-BED of the prior art.
Accuracy detection:
The AIDS new infections serum 12 confirmed through CDC is selected, previous infection serum 10 are pressed kit test method
It is detected, the HIV-Ag11 of HIV-BED and the HRP label of HRP label is selected to carry out Parallel testing.Accuracy testing result is asked
Referring to table 7.
The accuracy testing result of 7 two kinds of antigens of table
As shown in Table 7, the accuracy rate of HIV-Ag11 new infections accuracy rate ratio HIV-BED is high, HIV-Ag11 detection
Previous infection coincidence rate ratio HIV-BED high.
Detection of Stability:
Purified detected material HIV-Ag11 is divided into 2 parts, 1 part is placed in 4 DEG C of refrigerators, and another 1 part is placed in 37 DEG C
It takes out to be put into 4 DEG C of refrigerators after 6 days in insulating box and balance overnight, at detection plate, the ELISA Plate placed with 4 DEG C is closed coating with positive
Key quality-control product carries out Detection of Stability.Detection of Stability result please refers to table 8.
Remarks: coating reaction plate is to the detection A value of positive crucial quality-control product with 4 DEG C of ELISA Plates to positive crucial quality-control product
The ratio of detection A value is judged that ratio more high stability is better.
8 Detection of Stability result of table
As shown in Table 8, the stability for the recombinant antigen HIV-Ag11 that embodiment 3 provides is preferable.
To sum up, the embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections, the Recombinant antigen B
Amino acid sequence, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation, hydrophilic amino acid core sequence
And the lysine sequence of recombinant antigen both ends addition, hydrophilic amino acid core sequence are set to B, E, D amino acid sequence, BC
And between AE series of variation；B amino acid sequence as shown in SEQ ID No.1, E amino acid sequence as shown in SEQ ID No.2,
D amino acid sequence is as shown in SEQ ID No.3, and hydrophilic amino acid core sequence is as shown in SEQ ID No.4, BC series of variation
As shown in SEQ ID No.5, the AE series of variation is as shown in SEQ ID No.6.The solubility of the recombinant antigen is high, is easy to mention
It is pure, it is easy to mark, can be avoided or reduce the false positive reaction when detection of HIV new infections, improve the sensitivity and standard of detection
True property.
It include above-mentioned for detecting the nucleic acid of the recombinant antigen of HIV new infections the embodiment of the invention also includes having a kind of
Molecule, the expression vector including the nucleic acid molecules, the expression bacterial strain including the expression vector and including above-mentioned recombinant antigen or
The detection kit for being used to detect HIV new infections of nucleic acid molecules, the detection kit have the advantages that detection accuracy is high.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>newly create object Engineering Co., Ltd in Beijing
<120>a kind of recombinant antigen and its application for detecting HIV new infections
<130> 250
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 34
<212> PRT
<213>artificial sequence
<400> 1
Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln
1 5 10 15
Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Thr
20 25 30
Ala Pro
<210> 2
<211> 34
<212> PRT
<213>artificial sequence
<400> 2
Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Lys
1 5 10 15
Phe Leu Gly Leu Trp Gly Cys Ser Gly Lys Ile Ile Cys Thr Thr Ala
20 25 30
Ala Pro
<210> 3
<211> 32
<212> PRT
<213>artificial sequence
<400> 3
Leu Gln Ala Arg Ile Leu Ala Ile Glu Arg Tyr Leu Gln Asp Gln Gln
1 5 10 15
Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys His Ile Cys Thr Thr Thr
20 25 30
<210> 4
<211> 19
<212> PRT
<213>artificial sequence
<400> 4
Asp Lys Asp Ser Thr Gln Gln Asn Thr Gly Gly Pro Gln Thr Thr Ser
1 5 10 15
Lys Gly Gly
<210> 5
<211> 34
<212> PRT
<213>artificial sequence
<400> 5
Leu Gln Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln Gln
1 5 10 15
Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala
20 25 30
Val Pro
<210> 6
<211> 34
<212> PRT
<213>artificial sequence
<400> 6
Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Gln Asp Gln Lys
1 5 10 15
Phe Leu Gly Leu Trp Gly Cys Ser Gly Lys Ile Ile Cys Thr Thr Ala
20 25 30
Ala Pro
<210> 7
<211> 297
<212> PRT
<213>artificial sequence
<400> 7
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Gly Lys Lys Gly Lys Gly Ser Leu Gln Ala Arg
20 25 30
Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile
35 40 45
Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Thr Ala Pro Arg Gly
50 55 60
Asp Lys Asp Ser Thr Gln Gln Asn Thr Gly Gly Pro Gln Thr Thr Ser
65 70 75 80
Lys Gly Gly Lys Glu Phe Leu Gln Ala Arg Val Leu Ala Val Glu Arg
85 90 95
Tyr Leu Lys Asp Gln Lys Phe Leu Gly Leu Trp Gly Cys Ser Gly Lys
100 105 110
Ile Ile Cys Thr Thr Ala Ala Pro Arg Gly Asp Lys Asp Ser Thr Gln
115 120 125
Gln Asn Thr Gly Gly Pro Gln Thr Thr Ser Lys Gly Gly Val Asp Leu
130 135 140
Gln Ala Arg Ile Leu Ala Ile Glu Arg Tyr Leu Gln Asp Gln Gln Leu
145 150 155 160
Leu Gly Ile Trp Gly Cys Ser Gly Lys His Ile Cys Thr Thr Thr Arg
165 170 175
Gly Asp Lys Asp Ser Thr Gln Gln Asn Thr Gly Gly Pro Gln Thr Thr
180 185 190
Ser Lys Gly Gly Glu Phe Leu Gln Thr Arg Val Leu Ala Ile Glu Arg
195 200 205
Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys
210 215 220
Leu Ile Cys Thr Thr Ala Val Pro Arg Gly Asp Lys Asp Ser Thr Gln
225 230 235 240
Gln Asn Thr Gly Gly Pro Gln Thr Thr Ser Lys Gly Gly Val Asp Leu
245 250 255
Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Gln Asp Gln Lys Phe
260 265 270
Leu Gly Leu Trp Gly Cys Ser Gly Lys Ile Ile Cys Thr Thr Ala Ala
275 280 285
Pro Glu Phe Gly Gly Lys Lys Gly Lys
290 295
<210> 8
<211> 102
<212> DNA
<213>artificial sequence
<400> 8
ctgcaggcgc gcattctggc ggtggaacgc tatctgaaag atcagcagct gctgggcatt 60
tggggctgca gcggcaaact gatttgcacc accaccgcgc cg 102
<210> 9
<211> 102
<212> DNA
<213>artificial sequence
<400> 9
ctgcaggcgc gcgtgctggc ggtggaacgc tatctgaaag atcagaaatt tctgggcctg 60
tggggctgca gcggcaaaat tatttgcacc accgcggcgc cg 102
<210> 10
<211> 96
<212> DNA
<213>artificial sequence
<400> 10
ctgcaggcgc gcattctggc gattgaacgc tatctgcagg atcagcagct gctgggcatt 60
tggggctgca gcggcaaaca tatttgcacc accacc 96
<210> 11
<211> 57
<212> DNA
<213>artificial sequence
<400> 11
gataaagata gcacccagca gaacaccggc ggcccgcaga ccaccagcaa aggcggc 57
<210> 12
<211> 102
<212> DNA
<213>artificial sequence
<400> 12
ctccaaaccc gtgttctcgc gattgaacgt tacctgaaag accagcaact gctcggtatc 60
tggggttgct ctggcaaact catctgcact accgcagttc cg 102
<210> 13
<211> 102
<212> DNA
<213>artificial sequence
<400> 13
ctccaggcgc gtgtgctggc tgtcgagcgc tacctgcagg atcagaaatt cctcggtctc 60
tggggctgct ccggcaagat tatttgtacc actgcagcac ca 102
<210> 14
<211> 894
<212> DNA
<213>artificial sequence
<400> 14
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atgggcaaaa aaggcaaagg cagcctgcag gcgcgcattc tggcggtgga acgctatctg 120
aaagatcagc agctgctggg catttggggc tgcagcggca aactgatttg caccaccacc 180
gcgccgcgcg gcgataaaga tagcacccag cagaacaccg gcggcccgca gaccaccagc 240
aaaggcggca aagaatttct gcaggcgcgc gtgctggcgg tggaacgcta tctgaaagat 300
cagaaatttc tgggcctgtg gggctgcagc ggcaaaatta tttgcaccac cgcggcgccg 360
cgcggcgata aagatagcac ccagcagaac accggcggcc cgcagaccac cagcaaaggc 420
ggcgtggatc tgcaggcgcg cattctggcg attgaacgct atctgcagga tcagcagctg 480
ctgggcattt ggggctgcag cggcaaacat atttgcacca ccacccgcgg cgataaagat 540
agcacccagc agaacaccgg cggcccgcag accaccagca aaggcggcga attcctccaa 600
acccgtgttc tcgcgattga acgttacctg cgtgaccagc aactgctcgg tatctggggt 660
tgctctggcc gtctcatctg cactaccgca gttccgcgcg gcgataaaga tagcacccag 720
cagaacaccg gcggcccgca gaccaccagc aaaggcggcg tggatctcca ggcgcgtgtg 780
ctggctgtcg agcgctacct gaaggatcag cgtttcctcg gtctctgggg ctgctccggc 840
aagactattt gtaccactgc agcaccagaa tttggcggca aaaaaggcaa ataa 894
<210> 15
<211> 28
<212> DNA
<213>artificial sequence
<400> 15
cactataggg gaattgtgag cggataac 28
<210> 16
<211> 26
<212> DNA
<213>artificial sequence
<400> 16
ctcagcttcc tttcgggctt tgttag 26
Claims (10)
1. a kind of for detecting the recombinant antigen of HIV new infections, which is characterized in that it includes B amino acid sequence, E amino acid
Sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, the hydrophilic amino
Sour core sequence is set between B, E, D amino acid sequence, BC and AE series of variation；
The B amino acid sequence is as shown in SEQ ID No.1, and the E amino acid sequence is as shown in SEQ ID No.2, the D ammonia
Base acid sequence is as shown in SEQ ID No.3, and as shown in SEQ ID No.4, the BC becomes the hydrophilic amino acid core sequence
Different sequence is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.
2. according to claim 1 for detecting the recombinant antigen of HIV new infections, which is characterized in that described for examining
The N-terminal and C-terminal for surveying the recombinant antigen sequence of HIV new infections are added with lysine sequence that can be markup for improving antigen
Column.
3. according to claim 1 for detecting the recombinant antigen of HIV new infections, which is characterized in that in the recombination
In the sequence of antigen, the B amino acid sequence, E amino acid sequence, D amino acid sequence, the BC series of variation and AE variation sequence
Leie is arranged successively from the N-terminal of recombinant antigen to C-terminal.
4. according to claim 2 for detecting the recombinant antigen of HIV new infections, which is characterized in that the recombination is anti-
Former sequence is as shown in SEQ ID No.7.
5. a kind of described in any item for detecting the nucleic acid of the recombinant antigen of HIV new infections for encoding Claims 1 to 4
Molecule, which is characterized in that the nucleic acid molecules include B nucleic acid molecules, E nucleic acid molecules, D nucleic acid molecules, BC nucleic acid molecules, AE
Nucleic acid molecules and hydrophilic nucleotide sequence；
The nucleotide sequence of the B nucleic acid molecules is as shown in SEQ ID No.8, the nucleotide sequence of E nucleic acid molecules such as SEQ ID
Shown in No.9, the nucleotide sequence of D nucleic acid molecules is as shown in SEQ ID No.10, hydrophilic nucleotide sequence such as SEQ ID No.11
Shown, the nucleotide sequence of the BC nucleic acid molecules is as shown in SEQ ID No.12, the nucleotide sequence of the AE nucleic acid molecules
As shown in SEQ ID No.13.
6. according to claim 5 for detecting the nucleic acid molecules of the recombinant antigen of HIV new infections, which is characterized in that
The nucleotide sequence of the nucleic acid molecules is as shown in SEQ ID No.14.
7. a kind of expression vector, which is characterized in that it includes nucleic acid molecules described in claim 5 or 6；
Preferably, the expression vector is pET28a.
8. a kind of expression bacterial strain, which is characterized in that it contains expression vector as claimed in claim 7；
Preferably, the expression bacterial strain is E. coli expression strains.
9. expression bacterial strain according to claim 8, which is characterized in that the expression bacterial strain is e. coli bl21 (DE3).
10. a kind of for detecting the detection kit of HIV new infections, which is characterized in that it includes that Claims 1 to 4 is any
Recombinant antigen or described in claim 5 or 6 for detecting the nucleic acid molecules of the recombinant antigen of HIV new infections described in.
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| CN111187354A (en) * | 2020-02-20 | 2020-05-22 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit |
| CN113311159A (en) * | 2021-04-15 | 2021-08-27 | 南方医科大学 | Test strip for rapidly distinguishing recent and long-term HIV infection states through serum HIV-1 antibody detection and preparation method thereof |
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| CN113311159A (en) * | 2021-04-15 | 2021-08-27 | 南方医科大学 | Test strip for rapidly distinguishing recent and long-term HIV infection states through serum HIV-1 antibody detection and preparation method thereof |
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| CN110066343B (en) | 2021-04-09 |
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